Fermentation process for the production of l-ornithine

ABSTRACT

L-ornithine can be produced by cultivating an auxotrophic mutant of Escherichia coli which requires arginine or citrulline, but not ornithine for the growth in a cultivation medium containing assimilable carbon, nitrogen sources, inorganic ions and arginine or citrulline, phosphate ion of said medium being limited to about 0.5 - 3.5 Mu mole/ml, under aerobic condition.

United States Patent Ando et a], 51 June 6, 1972 [54] FERMENTATION PROCESS FOR THE [56] References Cited PRODUCTION OF L-ORNITHINE UNITED STATES PATENTS 2,988,489 6/1961 Kinoshita et al ..l95/47 Inventors: Kunio And, Kawasaki shi; mdeo oishi, 3,374,150 3/1968 Noguchi et a1 ..l95/47 Tokyo, both of Japan OTHER PUBLICATIONS [73] Assignee: Chugai Seiyaku Kabushiki Kaisha, Tokyo, Kinoshita article in Proceedings of the Fifth International 1 an Congress of Biochemistry, Moscow 1961, Vol. 8, pp. 43- 52 p (published 1963) [22] Filed: Jan. 2, 1969 Primary Examiner-Lionel M. Shapiro l A l. N 788,659 [2 1 pp 0 Attorney-Polachek&Saulsbury Related US. Application Data [57] ABSTRACT [63] Continuation-inart of 'Ser. No. 724,299, Feb. 5,

1968 abandone: which is a continuation of Ser No L-ornlthme can be produced by cultivating an auxotrophic 469 J 1 2 b d d mutant of Escherichia coli which requires arginine or citrulu y a an one line, but not omithine for the growth in a cultivation medium containing assimilable carbon, nitrogen sources, inorganic [52] US. Cl ..l95/29 ions and arginine or citmmne phosphate ion of Said medium [5] 1 13/06 being limited to about 0.5 3.5; mole/ml, under aerobic con- [58] Field of Search ..l95/29, 47

dition.

4 Claims, No Drawings FERlVlENTA TION PROCESS FOR THE PRODUCTION OF L-ORNITHINE This application isa continuation-in-part of our earlier filed copending application Ser. No. 724,299 filed on Feb. 5, 1968,

now abandoned said earlier application being a continuation of our first earlier filed application Ser. No. 469,307 filed July 2, 1965, now abandoned.

This invention relates to a fermentation process for the production process of L-ornithine and more particularly to the production of L-ornithine using an auxotrophic mutant of Escherichia coli.

It is well known that L-omithine plays an important role on the metabolism of the living body as a member of the ornithine cycle in the production of urea and has an excellent effect on the detoxication of ammonia.

Hitherto, L-ornithine has been produced by a fermentation method or a synthesizing method, mainly by the former using arginine or citrullinerequiring mutant of Micrococcus glutamicus known as glutarnic acid producing bacteria. The method using Micrococcus glutamicus is good for the production of L-ornithine, but yield is not only still so enough, but also the microorganism often requires biotin for the growth, different from the case of glutarnic acid formentation, or control of the pH of the medium during the fermentation by the addition of urea, and further it often requires the addition of surface active agent to the medium to produce L-ornithine.

An object of this invention is to provide an improved new fermentation process for the production of L-ornithine.

. Another object of this invention is to provide more efficacious and simple'fermentation process for the production of L-ornithine.

A further object of this invention is to provide a fermentation process for the production ofL-ornithine whereby bornithine can be obtained in high purity and good yield.

A still further object of this invention is to provide a fermentation process for the production of L-omithine whereby L-ornithine can be advantageously obtained on industrial scale.

This invention bases our new finding that certain auxotrophic mutants derived by ultraviolet irradiation from a stock culture strain of Escherichin coli accumulate a great amount of L-omithine in the medium, if it is cultivated in a cultivation medium containing assimilable carbon, nitrogen sources, inorganic ions and arginine or citrulline, phosphate ions of said medium being limited to about 0.5 3.5g. mole/ml. The auxotrophic mutant is strains which requires arg inine or citrulline, but not ornithine for the growth. One of the typical strain was deposited to American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 under the number of ATCC 21 104.

According to the present invention, L-ornithine can be obtained by (a) cultivating an auxotrophic mutant of Escherichia coli which requires arginine or citrulline, but not omithine for the growth in a cultivation medium containing assimilable carbon, nitrogen sources, inorganic ions and arginine or citrulline, phosphate ion of said medium being limited to about 0.5 3.5g. mole/ml, under aerobic condition; (b) accumulating L- omithine in said medium; and (c) recovering said L-ornithine. As for the medium components, ordinal assimilable carbon and nitrogen sources and ordinal inorganic salt may be used,

for example, there may be mentioned glucose, sucrose hydrolysate, starch hydrolysate and waste molasses hydrolysate as carbon source, meat extract, peptone, ammonium sulfate, ammonium chloride and corn steep liquor as nitrogen source, and as inorganic salts potassium hydrogen phosphate, fen-1c sulfate, ferric chloride andthe like may be used optionally at need.

Arginine or citrulline is to be added to the medium in a little less amount than that of best growth, preferably 0.3 1.0g. mole/ml, and it would rather be advantageous to use arginine than to use citrulline because arginine is cheaper than citrulline. This arginine may be replaced by the other natural substance which contains arginine, for example, peptone, meat extract, yeast extract, corn steep liquor, and protein hydrolysate, but it goes without saying that the concentration is to be a little less amount than that of best growth as the same in pure argmme. v v I Phosphate ion may be supplied with various organic and inorganic phosphates, for example, dipotassium phosphate.

The cultivation is carried out at a temperature from 28 to 32 C under aerobic condition,whereby L-ornithine is accumulated in the cultivation medium and accumulated L-or nithine may be separated purely bythe conventional method usually used for amino acid separation, for example, ion exchange resin treatment or concentration to syrup, after the removal of the cell by filtration or centrifugation. I

It has not been known that non-glutamic acid producing bacteriasuch as Escherichia coli accumulates Lornithine in the medium, if it is cultivated in a cultivation medium, phosphate ionconcentration being limited to a certain range andthe yield of L-omithine is over 35 percent (molar ratio) of glucose initially contained in the medium. The process of this invention is so eflicacious and simple that it is suitable to industrial scale production.

The following examples which are intended as infonnance and typical only and not in a limiting sense will further illustrate the invention. r

Example 1 An auxotrophic mutant Escherichia coli 8-19-19 (ATCC No. 21104) derived from the parent strain Escherichia coli B-l9 (stockculture of Inst. For' Infectious Diseases, The University of Tokyo) by ultraviolet irradiation method, which requires arginine or citrulline, but not omithine for the growth, was cultivated under shaking 'at 30 C for 96 hours using the following medium (pH: 6.0):

glucose 5% ammonium sulfate 1.5% dipotassium phosphate 0.025% or 0.1% magnesium sulfate 0.03% L-arginine hydrochloride 0.012% or 0.03% calcium carbonate The amounts of the cell (mg/ml) and the formed L-ornithine (mg/ml) were deter-mined and summarized in the following table:

(1n the table, the amount of L-ornithine is shown as that of hydrochloride salt).

As shown in the above table, it is most advantageous to use the medium containing 0.025 percent of dipotassium phosphate and 0.012 percent of arginine hydrochloride. Further, it was clarified that effect of dipotassium phosphate to the yield of L-ornithine was caused by phosphate ion and not by the potassium ion, because the same amount of L-ornithine was obtainedin case of cultivating the same bacteria in the same medium supplemented with 0.1% potassium chloride.

Example 2: V

To a sterilized cultivation medium (pH: 6.0) containing 5 percent of glucose, 15 percent of ammonium sulfate, 0.04 percent of dipotassium phosphate, 0.03 percent of magnesium sulfate (7 mole hydrate) and 0.01 percent of L-arginine auxotrophic mutant' Escherichia coli which is able to grow in the minimal medium containing either arginine or citrulline but notin the minimal medium containing omithine, which was derivedfromEscherichia coli 1AM 1222 (stock culture of Institute of. Applied Microbiology, The University of Tokyo) was inoculatedto 50 ml of the above cultivation medium poured aseptically into 500 ml-shitking flask and cultivated under. aerobic condition at 30 C for 96 hours. After completion of the cultivation, accumulated amount of L-omithine in the medium was determined to be 15 mg/ml (Yield ratio against consumed glucose added: 32 percent).

- After the clarification of the cultivation broth, L-omithine was isolated by ion exchange treatment and purified as hydrochloridecrystals. Yield: 0.6 gr. Example 3: g i

To a sterilized cultivation medium (pH: 0.6) containing 6 percent of glucose, 1.5 percent of ammonium sulfate, 0.04

" percent of dipotassium phosphate, 0.2 percent of corn steep liquor, 0.03 percent of magnesium sulfate (7 mole hydrate) and 0.012 percent of L-citrulline was added calcium carbonate which was separately sterilized by dry-heating to become 3 percent. An auxotrophic mutant Escherichia coli 8-19-19 (ATCC No. 21104), derived from the parent strain ATCC No. 21 104 which requires an amino acid selected from the group consisting of arginine and citrulline, but not or- Escherichia coli B-l9 (stock'culture of lnst for Infectious 4 nithine for the growth in a cultivation containing assimilable carbon, nitrogen sources, inorganic ions and an amino acid selected from the group consisting of arginine and citrulline, phosphate ion of said medium being limited to about 0.5 3.5 mole/ml under aerobic condition:(b) accumu-.

lating L-ornithine in said medium; and(c) recovering said L- ornithine. I

2. A process for the productionof bornithinelwhich comprises (a) cultivating an auxotrophic mutant Escherichia coli ATCC No. 21 104 which requires anamino acid'selected from the group consisting of arginine and citrulline, but not 0rnithine for the growth in a cultivation medium containing assimilable carbon, nitrogen sources, inorganic. ions and an amino acid selected from the group consistingof citrulline and arginine, phosphate ion and the amino acid of said medium' being a concentration of about 0.5 3.5g. mole/ml and 0.3

1.0 mole/ml, respectively, under aerobic condition; (b) accumulating L-omithine in said medium; and (c) recovering said L-ornithine.

3. A process for the production of I/ornithine which com- 9 prises (a) cultivating Escherichia coli ATCC No. 21104 in a cultivation medium containing assirnilable carbon, nitrogensources, inorganic ions and an amino acid selected from the group consisting of citrulline and arginine, phosphate ion of said medium being limited to about 0.5 3.5p molelml under aerobic condition: (b) accumulating L-ornithine in said mediumi and (c) recovering said L-ornithine.

4. A process for the production of L-ornithine which comprises (a) cultivating Escherichia coli ATCC. No. '21 104 in a cultivation medium containing assimilable carbon, nitrogen sources, inorganic ions and an aminoacid selected from the group consisting of citrullineand arginine, phosphate ion and the amino acid of said medium being a concentration of about 0.5 3.5;. mole/ml and 0.3 1.0;; mole/ml, respectively,- under aerobic condition; (b) accumulating L-ornithine in said medium; and (c) recovering said L-ornithine. 

2. A process for the production of L-ornithine which comprises (a) cultivating an auxotrophic mutant Escherichia coli ATCC No. 21104 which requires an amino acid selected from the group consisting of arginine and citrulline, but not ornithine for the growth in a cultivation medium containing assimilable carbon, nitrogen sources, inorganic ions and an amino acid selected from the group consisting of citrulline and arginine, phosphate ion and the amino acid of said medium being a concentration of about 0.5 - 3.5 Mu mole/ml and 0.3 - 1.0 Mu mole/ml, respectively, under aerobic condition; (b) accumulating L-ornithine in said medium; and (c) recovering said L-ornithine.
 3. A process for the production of L-ornithine which comprises (a) cultivating Escherichia coli ATCC No. 21104 in a cultivation medium containing assimilable carbon, nitrogen sources, inorganic ions and an amino acid selected from the group consisting of citrulline and arginine, phosphate ion of said medium being limited to about 0.5 - 3.5 Mu mole/ml under aerobic condition: (b) accumulating L-ornithine in said medium: and (c) recovering said L-ornithine.
 4. A process for the production of L-ornithine which comprises (a) cultivating Escherichia coli ATCC No. 21104 in a cultivation medium containing assimilable carbon, nitrogen sources, inorganic ions and an amino acid selected from the group consisting of citrulline and arginine, phosphate ion and the amino acid of said medium being a concentration of about 0.5 - 3.5 Mu mole/ml and 0.3 - 1.0 Mu mole/ml, respectively, under aerobic condition; (b) accumulating L-ornithine in said medium; and (c) recovering said L-ornithine. 